In nature, cytotoxic T-cells are responsible for identifying cells that display foreign antigens and killing them. However, T-cells do not only respond fully to typical foreign antigens but also show responses to self in the periphery in the form of co-agonist activity (wherein certain host self-ligands can synergize with agonist ligands in triggering the T-cell receptor complex to initiate signaling) or overt agonist activity in the case of autoimmunity or effective anti-tumor responses. One potential strategy is to compensate for ineffective anti- tumor responses by increasing TCR-CD8-peptide-MHC binding affinity. However, it is still not clear how much one can increase the potency of the system in order to mediate greater T-cell activity without mediating dangerous auto-reactivity to host antigens. Our long-term goal is to modify the T-cell activation threshold so that these immune effectors mediate efficient immune responses to disease-associated, self (tumor)-antigens while remaining unresponsive to the host's non-tumor associated self-antigens. The objective of the experiments proposed in this application is to make a major step towards this goal by determining the biophysical mechanism underlying how T-cells discriminate between different classes of self-ligands defined as tumor (self) and host (self) ligands. The central hypothesis of the application is that functional discrimination between self-ligands with co-agonist activity and those that mediate self-destructive responses is achieved by kinetic thresholding mediated by the conjoint action of the TCR and the CD8 co-receptor. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the quantitative relationship between TCR-CD8 binding avidity for specific peptide-MHC ligands and the co-agonist verses effector-inducing activity of these ligands;2) Determine the TCR affinity that defines the threshold for maintaining specificity to effector-inducing ligands;3) Determine how alterations in CD8 affinity will affect the ability of the T-cells to maintain discrimination among self-ligand classes. To achieve these aims we will use a well-established model system for recognition of "true" self-antigens involving expression of human self/tumor- reactive HLA-A2 restricted receptors on mouse CD8+ T-cells. We will use novel molecular fluorescent imaging and biophysical techniques to investigate the relationship between the biophysics of ligand recognition and functional responses. Finally, we will use in vitro phage display to select out high-affinity TCR and CD8 molecules which will allow us to access a larger kinetic window than available using naturally occurring TCR and CD8 molecules. The rationale for the proposed research is that a sufficiently large affinity window exists between normal tissue co-agonists and weak tumor agonists such that enhancing the conjoint TCR-CD8 avidity for the latter can improve effector responses without engendering undesirable anti-tissue responses. Relevance to human health: We believe this issue is important in understanding and enhancing cancer immunity and could lead to more effective therapies for this disease. Public Health Relevance: We expect that our studies will provide insight into how we can manipulate the immune response by increasing the affinity of the TCR-CD8-peptide-MHC interaction to mediate more effective recognition of tumor (self) antigens while avoiding self-reactivity. The knowledge obtained would be important in terms of generating high-affinity TCR or CD8 molecules that when transferred into CD8+ T-cells could be used for effective adoptive immunotherapy transfer therapies and still retain specificity. Alternatively, soluble TCRs with well-defined specificities could also be modified to deliver radionuclides, toxins or immunomodulatory molecules for more efficient killing of tumor cells.